Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis

Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-ribose polymers, which are involved in a wide range of cellular processes such as preservation of genome integrity, DNA damage signaling and repair, molecular switches between distinct cell death pathways, and cell cycle progression. Previously, we demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the parasite’s nucleolus. We also describe that TcPARP is present in a thread-like structure that connects two dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore, ADP-ribose polymers could also be detected in this thread during mitosis. These findings represent a first approach to new potential TcPARP functions inside the nucleus and will help understand its role well beyond the largely described DNA damage response protein in trypanosomatids.

further examination of more diverse role for PARylation in this parasite. Particularly as aspects of gene expression regulation, cell cycle control and DNA repair are unorthodox when compared to more "canonical" Eukaryotes.
We would like to thank reviewer 2 for critical reading of our manuscript and all comments that have helped us to improve the here revised version.
All suggested modifications, as well as the minor orthographic corrections, have been included in the present manuscript.

Major Concerns: This study is largely descriptive based on (at the face of it) a small number of images.
There is limited quantitative data to support the findings of the authors, just IFA images quantification has now been included in the manuscript and in the figure legend when appropriate.

There is no functional data relating to the localisation of TcPARP to the nucleolus.
I feel this may not be entirely necessarily for this study but would be nice to see.
They have a panel of overexpression mutants but there is very minimal characterisation of them despite some appearing to have slightly different growth patterns from the WT cell line (perhaps not significant but may be worth looking at in terms of DNA damage conditions etc) We made these strains expressing these constructs as a tool to better analyse the location of TcPARP in the nucleus and specially to describe the novel finding of its location in the thread that connects both nuclei in mitosis. We have also evaluated the growth curves of the different transgenic parasites under stress conditions (200 µM H 2 O 2 ) but we did not see significant differences between them. These results therefore seemed unattractive to include in the manuscript and we thought that it would not contribute to in the main point in our manuscript. However, we are sending the editor and reviewer these results (See Figure 1 below) for their consideration.
In the revised manuscript we have clarified which statistical test was used and the number of repetitions performed for the obtention of the results depicted in S1Fig. Error bars show the SEM of five independent experiments. A one-way ANOVA revealed no significant differences between the different growth curves.
Also, why not use the inhibitor to look at spindle formation/cytokinetic defeats etc?
Why not carefully profile the spindle progression across mitosis and the formation of the thread structure?
We appreciate the reviewer's interest in our work and we believe that these suggestions could be very valuable for future research. The performance of the suggested experiment presents several challenges: epimastigotes are very small compared to mammalian cells, so it is therefore difficult to get clear images of some cellular structures. Moreover, markers that allow the precise tracking of the different stages of mitosis are not readily available for this model. These facts difficult the possibility to make a precise profile of the cells progression across mitosis, even when working on synchronized cell cultures as we have previously done.
However, we performed a proof of concept with synchronized epimastigotes and observed the mitotic spindle in the G2-M phase of the cycle in parasites incubated both in the absence and in the presence of 100 nM Olaparib, a known PARP inhibitor. We were able to observe that the spindles are formed in both preparations without detecting differences. In any case, we believe that in order to obtain conclusions, better studies are required and further researches are needed. The results can be seen at FIGSHARE DOI 10.6084/m9.figshare.21108895.

Lack of reporting relating to data/experiments
 Basic descriptions missing relating to the data and methods  How many times an experiment has been performed?
 How the data has been processed?
 What the error bars represent?
 Some controls for IFA images etc?
 Type of microscope?
 Image processing pipeline?

 What if you are not seeing phenotypes simply due to poor overexpression of one truncated piece Vs anotherlooks like from the WB that the expression of the recombinant version of TcPARP is different in each cell line.
pRIBOTEX vector is not episomally maintained but integrated into a chromosome indistinguishable from the one encoding rRNA. It is a very useful tool since it allows more stable strains but with different degrees of expression, which cannot be easily regulated.
In this study we were interested in knowing if the parasite strains that express the different constructs had altered their growth capacity with respect to WT, but it was not our intention this time to study differences in other phenotypes of such parasite strains, as was stated before.

 Is there a negative (untagged) control/ vector control to account for nonspecific bands?
These controls are always performed, but they were not included in the figures to make them simpler. In any case, all the constructs have different molecular weights, which are reflected in the different patterns that do not present a common band attributable to the vector or something that suggest an unspecific binding of the antibody.
 What are the other bands on the blot? Several blots have additional bands PARP undergoes several post-translational modifications, of which the most important one is self-PARilation. We thought this is likely the case of TcPARP-FL, which contains all its domains. In this case, the upper band would be proposed for modified TcPARP, as this is a regularly pattern in these WBs.
We believe that in the case of delta NW and delta NRC partial degradation or incomplete transcription could have generated bands slightly smaller.
 I would recommend showing the complete WB uncut given the aforementioned?
We believe that for a better figure design the fragment shown is adequate.
 What ladder has been used?
We usually used SDS-PAGE -Prestained Protein Ladder -Blue Plus ® IV Protein Marker (10-180 kDa) Catalog Number：DM131-01-TransGen Biotech Co., LTD. We have incorporated this information to Materials and methods section.

SFig 1B & C  Looks like cell line delta-N is growing better than WT cells on a day-to-day basis?
The difference in growth rate is not statistically significant, according to figure 1 previously shown, nor did we see a different behaviour in the strain at longer times. Furthermore, we studied the cell doubling time for each strain and we did not find significant differences either (see  Our objective was to study the domain responsible for the localization of TcPARP in the nucleus and highlight the interesting finding that TcPARP and the PAR polymer are present in the nucleolus and in the thread in dividing nuclei. The different strains obtained expressing mutants were simply a useful tool for our studies and that is the reason because we decided include these results in Materials and Methods section, since we believe they could be useful for anyone else trying to replicate our experiments or conducting other evaluations.
Untagged control is showed in figure 1 and 4 upper panels.
 "White Field" -I think this should be "Bright Field"?
We apologize for the mistake; it has been corrected in all the figures.

 Number of replicates?
All the growth curve tests were carried out in three or more independent experiments, each one of them in technical triplicates. This statement was added to the legend figure.
 How was the cell body defined (i.e how the white dashed lines were generated)?
In the Merge quadrants some cells were contoured to illustrate the morphology of the parasites according to the bright field in the corresponding left panel. The dashed-line silhouette was then overlapped on the merge images using the ImageJ tools included in the ROI manager. The ImageJ free software was used for image processing.
We add the corresponding scale bar to the figures.

 What microscope were the cells captured on?
Samples were analyzed using an Olympus BX41 epifluorescence microscope.
We added this information in Materials and Methods section.

SFig. 3:
I would replace the term overexpressant. "TcVsp34 overexpression" (See comments above also which apply to this figure) Figure and legend were changed as suggested.

Figures (Main)
Comments again apply to all figures:

1) Bright Field?
This has been corrected in all figures.

2) Scale bars -I would put on each Bright Field image as it cannot be assumed they are all on the same scale
Scale bars have been added to all figures.

3) How many times was this experiment performed?
a. Images are representative of?

4) How are the images processed?
Control images without primary antibodies were taken in an analogous condition, during the same microscopy session. All images in each experimental series were taken with the same setting at the same microscope session. If modified, all were subjected to the same degree of brightness/contrast adjustment, including the control without a primary antibody. The ImageJ free software was used for confocal image processing and JaCoP Fiji's plugin was used for colocalization analysis. We have incorporated this clarification in the Materials and methods section. Number of experiment was introduced in the legend figures.

a. What microscope was used?
Samples were analyzed using an Olympus BX41 epifluorescence microscope or a Leica SPE confocal microscope (see Materials and Methods).

5) Quantification? Co-localisation plots of signal in Image J?
The Image J program was used for both fluorescence intensity measurement and confocal microscope image processing. For the co-localization assay, the JaCoP (Image J) tool was used. The signal from two channels (red and green) was analyzed in each single epimastigote.
Co-location coefficients were incorporated into the text or figure legends when appropriate.

6) Are all the cells in mitosis at the same stage of mitosis? In some cases for
instance the TcPARP delta WRC has a thicker appearance (Fig. 3, 4) vs much thinner in Fig. 2? The cultures were not synchronized, however from the images we assume that the stages analyzed were a late stage in mitosis, as is discussed in the Discussion section.

7) Some bright field images are missing
We have included the bright field only in the first figures to show readers outside the field the epimastigote morphology. Addition of an extra panel with bright field would oblige to reduce other images with loss of information.

Comments:
Line 134: re-localise from where? This needs to be stated The sentence was clarified.

Line 134: "These" results
The mistake in line 143 was corrected.

Line 135: "different arrangements" you need to define this betterwhat are you aiming to dorefer to the supplementary figure here and not in the methods.
We agree with the referee's observation, hence supplementary figure S1 is now mentioned in Results section.
Line 145: the use of the word "notoriously" doesn"t fit in the sentence. Do you mean consistently or?
The term notoriously was replaced by "particularly", since we want to highlight this new observation compared to what was previously described for TcPARP localization. Lines 157-160:

State again which truncated versions? Only those that have N-terminal sections?
This paragraph was clarified, as suggested.

You are suggesting that the reason you see the mis-localisation is due these cells being in stationary.
 You want to look at dividing cells but are you collecting cells too far into stationary phase and this is why you are seeing this?
The reviewer observation is pertinent. As we stated in the text, we describe this observation only in some cells. Our experiments are performed with cultures on day four after subculturing, which corresponds to an exponential growth stage. Our reference to stationary cells pointed to previous observations by other researchers which we do not believe are the reason for the observation in our case. We change this statement in the text to try to clarify this.
 Do you see multiple foci as reported by Gluenz? They say they see many foci of the antibody in stationary cells.
Even though the staining was not uniform we were not able to define them as Gluenz does in her work. As was stated before, our redaction was not clear so it was changed to avoid misleading.
 Is the nucleolar staining found in every cell? you say that that is not the case, and some is found in the nucleoplasm? How many cells in the population?

 How does this relate to the cell cycle given the nucleolus is consistent across all cell cycle stages? Is there always PARP signal associated with the nucleolus?
We have not carried out studies across all cell cycle stages, but we agree with the reviewer that it would be interesting to study it in the future.
Line 165-you mean of your overexpressed construct? I guess stationary phase epis would have reduced transcription relative to log phase.
As indicated before, this statement was rewritten for clarification.
Line 169: this is not quantified so how are you making this statement?
We measured the average area corresponding to the nucleolus for the epimastigotes in the different experimental conditions by using Image J software and found the following results: 0.00194 ± 0.00132 (n=29) for WT, 0.00233± 0.00217 (n=58) for 3AB, 0.00187± 0.00281 (n=32) for Olaparib, 0.00103±0.00149 (n=154) for Vps 34 overexpressors and 0.00059±0.00061 (n=59) for Overexpressors + CXH treated parasites. A high dispersion associated to the data rendered the differences as not statistically significant. We have modified the paragraph accordingly.

Line 178: "Scale bar" not just bar
It was corrected.

TcPARP is present in a connecting wire between nuclei of dividing epimastigotes
Generally: I would choose either "wire" or "thread" when referring to the localisation.
Thread was chosen to describe the thin structure that joins the dividing nuclei and the term wire has been removed from the manuscript. We have only localized L1C6 nucleolar marker. The sentence was rewritten for clarification.

Line 186: the cores of what?
The term "cores" was replaced by "nuclei" Line 185: Again, what nucleolar proteins? L1C6 is but tubulin is not just a nucleolar protein?
The sentence was reformed. A thinner thread was not always observed for the truncated versions (see Fig 4).
Although we agree with the hypothesis that this is dependent on the mitosis stage, as we worked with non-synchronic cultures, identifying a large number of dividing cells was difficult.
Legend and other mistakes were corrected.

PARylated proteins are present in the connecting thread
Line 204 You should elaborate here more about PAR and the assay you will use for non-expertsjust a sentence to help.
A sentence was added as suggested and the paragraph is now clearer.
Line 207: How do your over expressors respond to genotoxic stressmore sensitive or less?? Do they produce more PAR (how is this quantified?) Would be good to know if they produce more-or-less relative to WT cells for example.
We have quantified the amount of polymer present in the nuclei of parasites over expressing different constructs by using ImageJ software. A representative image of how the over expressors behave when challenged with H 2 O 2 (200 uM) is shown in figure 3 of this text and reviewers can find a table with the processed data. We could not conclude if they produce significant different amount of PAR in oxidative stress condition because of the high dispersion of the data.  We have previously demonstrated (PloS One 2012) that PARP-specific inhibitors were capable of inhibiting the proliferation of T. cruzi epimastigotes in nanomolar concentrations. As it was said before, we believe that it would be very interesting for our future publications to deeply study whether spindle alterations are produced by these inhibitors.
Line 245: why again not investigate this in more detail?
We agree with the reviewer in that it would be interesting to further investigate this phenomenon in the future. However, we consider that it is worth communicating the results already obtained to the research community as they might encourage colleagues working on this topic in trypanosomatids or other parasites.